4, 8, 14-trimethyl-18-nor-8alpha, 9beta, 14beta-androst-4-ene-3, 17-dione



United States Patent 3,351,6404,8,14-TRlMETHYL-18-NQR-8a,9fl,14fl-ANDROST- 4-ENE-3,17-DIONE Patrick A.Diassi, Westfield, and Pacifico A. Principe, South River, N.J.,assignors to E. R. Squibb & Sons,

Inc New York, N.Y., a corporation of Delaware No Drawing. Filed Mar. 23,1966, Ser. No. 536,623 3 Claims. (Cl. 260--397.3)

This invention relates to and has for its object the provision of newphysiologically active compounds, having the formula The novel compoundsof this invention are pharmacologically active substances which possessanti-androgenic activity (i.e., they inhibit the actions of androgens),and which may be used in the treatment of such conditions ashyperandrogenic acne. They also possess anti-estrogenic and androgenicactivity.

The compounds may be formulated for such administration, theconcentration and/or dosage being based on the activity of theparticular compound and the requirements of the patient.

The final product of this invention is prepared by the process of thisinvention which entails beginning with 4a,8,l4-trimethyl-18-nor50,8oc,9fi,l4,8-aI1ClIOSl3n-3,l7- dione and 40:,8,l4trimethyl-l8-nor-5a,8a,9,B,13a,14fi-androstane-3,l7-dione as startingmaterial. The preparation of these compounds is disclosed in copendingUS. patent application, Ser. No. 406,877, filed October 27, 1964. It hasbeen found that the compounds of this invention can be prepared from thestarting material by subjecting the latter to the action of amicroorganism of the genus Pseudomonas or to the action of the enzymesthereof under oxidizing and preferably aerobic conditions.

To prepare the compounds of this invention, the starting material may befirst subjected to the action of enzymes of a microorganism of the genusPseudomonas under oxidizing conditions. This oxidation can best beeffected either by including the starting material in an aerobic cultureof the microorganism, or by bringing together in an aqueous medium, thecompounds, air, and enzymes of nonproliferating cells of themicroorganism.

In general, the conditions of culturing the Pseudomonas microorganismfor the purposes of this invention are (except for the inclusion of thestarting material to be converted), the same as those of culturingvarious other microorganisms. As, for example, for the production ofantibiotics and other like substances. The microorganism is grownaerobically in contact with (in or on) suitable fermentation medium. Asuitable medium essentially comprises a source of carbon and energy. Thelatter may be a carbohydrate, for example, molasses, glucose, maltose,starch or dextrin, a fatty acid, a fat and/or the compound itself.Preferably, however, the medium includes an assimilable source of carbonand energy in addition to the steroid. Among the fats utilizable for thepurpose of this invention are lard oil, soybean oil, linseed oil,cottonseed oil, peanut oil, coconut oil, corn oil, castor oil, sesameoil, crude palm oil, fancy mutton tallow, sperm oil, olive oil,tristearin, tripalmitin, triolein and trilaurin. Among the fatty acidsutilizable for the purpose of this invention are stearic acid, palmiticacid, oleic acid, linoleic acid and myristic acid.

The source of nitrogenous factors utilizable for the purposes of thisinvention may be organic (e.g., soybean meal, cornsteep liquor, yeastextract, meat extract and/ or distillers solubles) or synthetic (i.e.,composes of simple, synthesizable organic or inorganic compounds, suchas ammonium salts, alkali nitrates, amino acids or urea).

An adequate sterile air supply should be maintained during fermentation,for example, by the conventional methods of exposing a large surface ofthe medium to air or by utilizing submerged aerated culture. Thec0mpound may be added to the culture during the incubation period, orincluded in the medium prior to sterilization or inoculation. Thepreferred (but not limiting) range of the concentration of the compoundin the culture is about 0.01% to about 0.1%. The culture period (orrather the time of subjecting the compound to the action of the enzyme)may vary considerably, the range of about twenty-four to ninety-sixhours being feasible, but not limiting.

The microbial process described hereinabove yields4,8,l4-trimethyl-18-nor-8a,9,8,14B-androst 4 ens-3,17- dione and4,8,14-trimethyl-18-nor-8a,9;3,l3a,l4/3-androst- 4-ene-3,l7-dione.

The invention may be illustrated by the following examples, alltemperatures are in degrees centigrade unless otherwise stated:

Type Culture Collection) No. 11996, the slants containing as a nutrientmedium (A):

Grams Glucose l0 Yeast extract 2.5 K HPO 1 Agar 2O Distilled water to 1liter.

is suspended in 5 ml. of 0.01% aqueous sodium lauryl sulfate solution.One ml. portions of this suspension are used to inoculate sixteen 250ml. Erlenmeyer flasks, each containing 50 ml. of the followingsterilized medium Grams Beef extract 1.5 Yeast extract 3 Peptone 6Dextrose 1 Distilled water to 1 liter.

After eighteen hours incubatiton at 25 C. with continuous rotaryagitation (280 cycles/minute; two-inch radius), 5% (v.:v.) transfers aremade to one hundred 250 ml. Erlenmeyer flasks, each containing 50 ml. offreshly sterilized medium B. After twenty-four hours of furtherincubation, using the same conditions described above, each flask issupplemented with 200 micrograms/ ,ml. of 4a,8,14-'trimethyl18-nor-5ot,ltx,9[i,14fi -and'rostane-3,17-dione. The steroid is added bysupplementing each flask with 0.25 ml. of a sterile solution (40mg./ml.) of the steroid in N,N-dimethylformamide. A total of 1.0 gm. isfermented. After six days of further incubation, using the sameconditions as described above, the contents of the flasks are pooled andthe broth is then adjusted to pH 4.0 using 12 N H The acidified broth isthen filtered through a Seitz clarifying pad. The flasks mycelium andpad are washed with successive 50 ml. portions of warm water. Thecombined filtrate and washings have a volume of 5700 ml. They areextracted three times with 1900 ml. portions of chloroform which arecombined, washed twice with 2 liter portions of water and evaporated, invacuo. The residue is plate chromatographed on Woelm neutral alumina(Activity V) using chloroform as the developing solvent. The band atRfzQS is eluted with ethyl acetate, evaporate and crystallized fromacetone-hexane to give 4,8,14-trimethyl- 18 nor 8a,95,145androst-4-ene-3,17 dione having a melting point about 210-212 C., [a]+254 (chloroform),

A313, 254 m, (6, 14,400), max.

T g gi 8.26 (5., 4-011 8.65 5., CH 8.79 (S., CH 9.27 (s., CH

A rzalysis.Calcd for C H O (314.45): C, 80.21; H, 9.62. Found: C, 80.39;H, 9.47.

Following the procedure of Example 1 but substituting4a,8,14-trimethyl-l8-nor-5u,8ot,9B,13a,l4B androstane- 3,17-dione forthe 40:,8,14-trimethyl-18-nor-5a,8a,9fl,14B- androstane-3,17-dione thereis obtained 4,8,l4-trimethyl-18Il0l"-8a,9fl,l3oc,l4B-E1l'ldl'OSt-4-Bl'18-3,l7-diOn6.

The invention may be variously otherwise embodied within the scope ofthe appended claims.

1743, 1668, 1621 (MIL-1,

4 What is claimed is: 1. A compound having the formula o H II 5 2. -Acompound in accordance with claim 1 having the name4,8,14-trimethyl-18-nor-8a,9fl,145-androst-4- ene-3,17-dione.

3. A compound in accordance with claim 1 having the name4,8,l4-trimethyl-18-nor-8a,9fl,l3a,l4fi-androst- 4-ene-3,l7-dione.

20 References Cited UNITED STATES PATENTS 3,274,219 9/1966 Krakower260397.45

OTHER REFERENCES Comptes Rendus, vol. 258, pages 3491 to 3494 (Apr. 1,1964).

Experientia, vol. 20 (1964), pages 344 to 347.

ELBERT L. ROBERTS, Primary Examiner.

1. A COMPOUND HAVING THE FORMULA